A Fluorescence Dequenching-based Liposome Leakage Assay to Measure Membrane Permeabilization by Pore-forming Proteins.

Pore-forming toxins (PFTs) have been discovered in a wide range of organisms. Their functions are essential to the survival or virulence of many species. PFTs often interact with lipid membranes. Large unilamellar vesicles (LUV), also known as liposomes, have been commonly used as reliable membrane models for testing PFTs activity. Liposomes have great adaptability in size, lipid composition, and loading cargo. Incorporating the fluorescent dye/quencher pair, 8-Aminonaphthalene-1,3,6-Trisulfonic Acid (ANTS) and p-Xylene-Bis-Pyridinium Bromide (DPX), in liposomes is an effective approach for measuring membrane leakage. When ANTS and DPX are encapsulated in a liposome, the fluorescence of ANTS is quenched by DPX. However, disruption of liposome integrity and subsequent leakage result in measurable fluorescence emitted by ANTS. Here, we report our protocol for optimal liposome preparation for measuring liposome leakage by fluorescence dequenching.